Amplification of nucleic acid sequences has become a very important technology in the biological sciences. The best known of these techniques is the polymerase chain reaction (PCR). This procedure amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of oligonucleotide primers, and extension of the primers with DNA polymerase (Mullis KB et al., U.S. Pat. Nos. 4,683,202, 4,683,195; Mullis K. B., EP 201,184; Erlich H., EP 50,424, EP 84,796, EP 258,017, EP 237,362; Erlich H., U.S. Pat. No. 4,582,788; Saiki R. et al., U.S. Pat. No. 4,683,202; Mullis K. B. et al. (1986) in Cold Spring Harbor Symp. Quant. Biol. 51:263; Saiki R. et al. (1985) Science 230:1350; Saiki R. et al. (1988) Science 231:487; Loh E. Y. et al. (1988) Science 243:217; etc.). (All references cited herein are hereby incorporated by reference.) These steps can be repeated many times, potentially resulting in large amplifications of the number of copies of the original specific sequence. It has been shown that even single molecules of DNA can be amplified to produce hundreds of nanograms of product (Li H. et al. (1988) Nature 335:414). Though PCR is widely used, it has several technical weaknesses, including need for a specialized piece of equipment and the amount of time needed to perform all of the cycles necessary to attain the desired level of amplification.
Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh D. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Gingeras T. R. et al., PCT appl. WO 88/10315 (priority: U.S. patent application Ser. Nos. 064,141 and 202,978)). Schemes based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, are also known (Wu D. Y. and Wallace R. B. (1989) Genomics 4:560).
Miller H. I. and Johnston S., PCT appl. WO 89/06700 (priority: U.S. patent application Ser. No. 146,462, filed Jan. 21, 1988), disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (ssDNA) followed by transcription of many RNA copies of the sequence. This scheme was not cyclic; i.e. new templates were not produced from the resultant RNA transcripts.
Davey C. and Malek L. T., EP 0,329,822, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (ssRNA), ssDNA, and double-stranded DNA (dsDNA). The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5'- to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a dsDNA molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
All of the above amplification procedures depend on the principle that an end product of a cycle is functionally identical to a starting material. Thus, by repeating cycles, the nucleic acid is amplified exponentially.
Methods that use thermo-cycling, e.g. PCR or Wu and Wallace, supra. have a theoretical maximum increase of product of 2-fold per cycle, because in each cycle a single product is made from each template. In practice, the exponent is always lower than 2. Further slowing the amplification is the time spent in changing the temperature. Also adding delay is the need to allow enough time in a cycle for all molecules to have finished a step. Molecules that finish a step quickly must "wait" for their slower counterparts to finish before proceeding to the next step in the cycle; to shorten the cycle time would lead to skipping of one cycle by the "slower" molecules, leading to a lower exponent of amplification.
Methods that include a transcription step, e.g. that of the present invention or of Davey and Malek, supra. can increase product by more than a factor of 2 at each cycle. As 100 or more transcripts can be made from a single template, factors of 100 or more are theoretically readily attainable. Furthermore, if all steps are performed under identical conditions, no molecule finishing step need "wait" for any other before proceeding to the next step. Thus amplifications that are based on transcription and that do not require thermo-cycling are potentially much faster than thermo-cycling amplifications such as PCR.